JOURNAL OF BACrERIOLOGY, Sept. 1992, p. 5669-5675 0021-9193/92/175669-07$02.00/0
Vol. 174, No. 17
Copyright © 1992, American Society for Microbiology
Characterization of Aromatic Dehalogenases of Mycobacterium fortuitum CG-2 J. S. UOTILA,1* V. H. KITUNEN,2 T. SAASTAMOINEN,l T. COOTE,' M. M. HAGGBLOM,3 AND M. S. SALKINOJA-SALONEN4 Department of General Microbiology, University of Helsinki Mannerheimintie 172, SF-00300 Helsinki, Finnish Forest Research Institute, SF-01301 Vantaa,2 and Research and Development Activities, Environmental Unit, University of Helsinki, SF-iSiO Lahti,4 Finland, and AgBiotech Center, Cook College, Rutgers University, New Brunswick, New Jersey 08903-20313 Received 28 February 1992/Accepted 22 June 1992
Two different dehalogenation enzymes were found in cell extracts of Mycobacteriumfortuitum CG-2. The first enzyme was a halophenol para-hydroxylase, a membrane-associated monooxygenase that required molecular oxygen and catalyzed the para-hydroxylation and dehalogenation of chlorinated, fluorinated, and brominated phenols to the corresponding halogenated hydroquinones. The membrane preparation with this activity was inhibited by cytochrome P-450 inhibitors and also showed an increase in the A4,, caused by CO. The second enzyme hydroxylated and reductively dehalogenated tetrahalohydroquinones to 1,2,4-trihydroxybenzene. This halohydroquinone-dehalogenating enzyme was soluble, did not require oxygen, and was not inhibited by cytochrome P-450 inhibitors.
Actinomycetes are a large and variable group of microorganisms occurring in many habitats, where they take part in the degradation of organic material. Many microbial strains degrading various aromatic pollutants have been isolated from this group. Enzymes so far characterized have been reported to be monooxygenases, hydroxylases, or dioxygenases (for a review, see reference 31). Actinomycetes, including those degrading xenobiotic compounds, have also been shown to be environmentally tenacious, making them a potential source of microorganisms for bioremediation (6, 18, 29, 30). Mycobacterium fortuitum CG-2 was isolated from a tetrachloroguaiacol enrichment culture and shown to degrade several chlorinated phenols, guaiacols, and syringols at micromolar levels (15). Pentachlorophenol (PCP) was shown to be mineralized to CO2, and tetrachloro-para-hydroquinone (TeCH) was detected as an initial intermediate. We have shown that the degradation of polychlorophenols, guaiacols, and syringols proceeds through chlorinatedparahydroquinones in three different Rhodococcus chlorophenolicus strains (1, 3, 12, 13, 15) and is mediated by a membrane-associated cytochrome P-450 enzyme (28). The product of this para-hydroxylation, TeCH, is further orthohydroxylated by a soluble halohydroquinone dehalogenase (4, 14, 28). In this paper, we report on the enzymatic dehalogenation of chlorinated, brominated, and fluorinated phenols and hydroquinones by M. fortuitum CG-2 cell fractions. We show that the halogenated phenols were converted to halogenated para-hydroquinones by a membrane-associated cytochrome P-450 enzyme. The halohydroquinones formed by the para-hydroxylation were further dehalogenated by a soluble halohydroquinone dehalogenase. The results show that M. fortuitum CG-2 dehalogenases are similar to those previously described by us for R. chlorophenolicus PCP-1 and CP-2 (3, 4, 14, 15, 28).
*
MATERIALS AND METHODS Bacterial strains and culture conditions. M. fortuitum CG-2 (12, 16) was grown in nutrient broth-yeast extract medium (21) in a gyratory shaker at 28°C. Two-day-old cultures were harvested or induced by the addition of increasing amounts (10 to 50 FtM) of PCP at 24-h intervals and then harvested. Cell fractionation. Cells were harvested by centrifugation (3,000 x g, 10 min, 4°C), resuspended (1 g [wet weight] ml-') in borax buffer (50 mM, pH 8.0) (22), and washed once with the same buffer. Cells were disrupted in a French press at 700 lb/in2, DNase I (50 ,g ml-'; Boehringer Mannheim, Mannheim, Germany) was added to the cell extract, and the mixture was incubated for 1 h at 22°C. The unbroken cells were removed by centrifugation (3,000 x g, 10 min, 4°C), and the supernatant was further centrifuged at 150,000 x g for 90 min at 4°C. The supernatant contained the soluble proteins, and the pellet contained the membrane-associated proteins. The pellet was washed once with 10 volumes of borax buffer and resuspended in the original volume of the same buffer, Analyses. For activity assays, 1.0 mg of protein ml-' and 20 to 50 ,uM substrate (PCP, pentafluorophenol [PFP], pentabromophenol [PBP], TeCH, tetrafluoro-para-hydroquinone [TeFH], or tetrabromo-para-hydroquinone [TeBH]) were used. Protein was determined by the method of Bradford (5) with a reagent from Bio-Rad Laboratories, Richmond, Calif., and with egg white lysozyme (Sigma, St. Louis, Mo.) for calibration. Assays for halophenol dehalogenation were carried out in borax buffer at pH 8.0. The pH optimum of the reaction was tested with borax buffer (pH range, 5.8 to 9.2). For assays for halohydroquinone dehalogenation, ascorbic acid (final concentration, 1 mg ml-') was added to prevent abiotic oxidation of halohydroquinones. The enzymatic activity was calculated from the linear period of activity (4 h). A reaction mixture with no enzyme added served as the blank for both types of assays. Halogenated phenolic compounds were analyzed as acetylated derivatives by gas-liquid chromatography (GLC) by an internal standard method as described earlier (2, 3). To improve the acetylation efficiency of the brominated conge-
Corresponding author. 5669
5670
UOTILA ET AL.
ners, we added acetone to the acetylation buffer to 10% (2). Fluorinated congeners were extracted in pentane, and brominated and chlorinated congeners were extracted in heptane. An HP-5 fused silica capillary column (25 m by 0.2 mm; 0.33 ,um; Hewlett Packard, Palo Alto, Calif.) was used. The temperature programs used for the analysis were as follows: for TeCH and TeBH, 50°C for 1 min and 10°C/min to 300°C; for TeFH, 20°C for 1 min, 10°C/min to 60°C, and 50°C/min to 180°C. Metabolites emerging were identified by GLC-mass spectrometry (MS) as described by Apajalahti and SalkinojaSalonen (3, 4). In the analysis of para-hydroxylation products with an 1802 label, the following ions were used:
2,3,5-trichloro-para-hydroquinone (2,3,5-TCH), mle 212, 214, 216, 218, 220. Anoxic assays and isotopic labeling. For the anoxic assays, the reaction mixture was enclosed in a gas-tight vial and oxygen was removed by sequential evacuation and purging with argon (99.998%). A similarly deoxygenated substrate was injected into the vial to start the reaction, and acetylation reagents were injected to stop the reaction. In the oxygen labeling experiments, 1802 gas (isotopic purity, 80 to 90%; Msd isotopes, Montreal, Quebec, Canada) was injected into the ampoule to a partial 1802 pressure of 0.1 and the reaction was started by the injection of a deoxygenated substrate (2,3,5-trichlorophenol [2,3,5-TCP]) into the ampoule to a final concentration of 20 ,uM and stopped by the addition of acetylation reagents after 4 h of incubation at 22°C. Labeling experiments with H2180 were performed like the aerobic ones, except that the solution was made 20% with respect to H2180-labeled water (isotopic purity, 97%; Aldrich Chemical Co., Milwaukee, Wis.). The reaction was started by the addition of an anaerobic substrate solution (2,3,5-TCP; final concentration, 20 ,uM). Inhibition assays. The inhibitors tested were Tiron (5 mM; Siegfried S.A.), methylenebisthiocyanate (5 mM; Riedel-deHaen), metyrapone (5 mM; Sigma), menadione (5 mM; vitamin K3, prereduced; Sigma), parathion (5 mM; Ehrenstorfer, Augsburg, Germany) and SKF-525A (5 mM; Smith Kline & French Laboratories). Inhibition by carbon monoxide was tested at partial pressures of 1.0, 0.1, and 0.01 in gas-tight vials. Spectrophotometric analysis. Absorption spectra were measured at room temperature with the single-beam mode of a Shimadzu UV-3000 spectrophotometer. Sodium dithionite (a few grains) was added to the protein solution (2.3 mg in 0.5 ml of borax buffer) to create a reduced environment, and the spectrum (400 to 500 nm) was measured before and after purging with carbon monoxide. The concentration of cytochrome P-450 was calculated from the reduced carbon monoxide differential spectrum with the specific extinction coefficient of the Pseudomonas putida enzyme, A45049 = 92.8 cm-' mM-1 (11). RESULTS Localization of the halophenol-para-hydroxylating activity in cell extracts of M. fortuitum CG-2. From previous work, we knew that M. fortuitum CG-2 was able to degrade (mineralize) PCP via para-hydroxylation to TeCH (15). To test whether other pentahalophenols would be attacked, we tested the degradation of PFP and PBP by crude extracts of PCP-induced cells and analyzed the products formed from PEP and PBP by GLC-MS. A new peak emerged in the gas chromatogram of each halophenol after incubation with the crude extract, the retention times (the elution times, in minutes, of the model compounds TeCH, TeBH, and TeFH
J. BACTERIOL.
were 11.3, 16.1, and 8.1, respectively, and those for the
metabolites formed from these pentahalogenated phenols were the same) and the mass spectra (Fig. 1) being identical to those of authentic TeCH, TeFH, and TeBH. These results
indicate that the pentahalophenols had undergone dehalogenation and para-hydroxylation while in contact with the crude extracts of M. fortuitum CG-2. The reaction was optimal at pH 7 to 8. Outside the pH range of 6.5 to 9.0, no dehalogenation was observed. To localize the PCP-, PFP-, and PBP-para-hydroxylating activity in M. fortuitum CG-2 cells, we prepared cell-free crude extracts from the cells and separated them into a 150,000 x g supernatant (soluble proteins) and a 150,000 x g pellet (membrane-associated proteins). Dehalogenation of halophenols was tested for each fraction as described in Materials and Methods. Table 1 shows the dehalogenation activities of the extracts prepared from PCP-induced and uninduced cells of M. fortuitum CG-2 and the localization of the halophenol-consuming enzyme activity in the cell fractions. The rates of turnover of PCP, PFP, and PBP in crude extracts from PCP-induced cells ranged from 1.7 to 2 nmol h-1 mg of protein-1 (Table 1). Crude extracts prepared from uninduced cells showed no detectable halophenol-degrading enzyme activity, indicating that this enzyme activity was inducible. Treatment of the crude extracts with proteinase K (1 mg ml-') destroyed the halophenol-para-hydroxylating activity, showing that it was mediated by enzyme protein. The induction factor for the halophenol dehalogenases was ca. 40-fold in M. fortuitum CG-2. When the cell extracts were fractionated by centrifugation (150,000 x g), 67% of the halophenol-degrading enzyme activity of the crude extracts was recovered in the 150,000 x g pellet, which contained 33% of the total cell extract protein (Table 1). No halophenol-para-hydroxylating activity was found in the 150,000 x g supernatant. Phenol was not degraded (
Vol. 174, No. 17
Copyright © 1992, American Society for Microbiology
Characterization of Aromatic Dehalogenases of Mycobacterium fortuitum CG-2 J. S. UOTILA,1* V. H. KITUNEN,2 T. SAASTAMOINEN,l T. COOTE,' M. M. HAGGBLOM,3 AND M. S. SALKINOJA-SALONEN4 Department of General Microbiology, University of Helsinki Mannerheimintie 172, SF-00300 Helsinki, Finnish Forest Research Institute, SF-01301 Vantaa,2 and Research and Development Activities, Environmental Unit, University of Helsinki, SF-iSiO Lahti,4 Finland, and AgBiotech Center, Cook College, Rutgers University, New Brunswick, New Jersey 08903-20313 Received 28 February 1992/Accepted 22 June 1992
Two different dehalogenation enzymes were found in cell extracts of Mycobacteriumfortuitum CG-2. The first enzyme was a halophenol para-hydroxylase, a membrane-associated monooxygenase that required molecular oxygen and catalyzed the para-hydroxylation and dehalogenation of chlorinated, fluorinated, and brominated phenols to the corresponding halogenated hydroquinones. The membrane preparation with this activity was inhibited by cytochrome P-450 inhibitors and also showed an increase in the A4,, caused by CO. The second enzyme hydroxylated and reductively dehalogenated tetrahalohydroquinones to 1,2,4-trihydroxybenzene. This halohydroquinone-dehalogenating enzyme was soluble, did not require oxygen, and was not inhibited by cytochrome P-450 inhibitors.
Actinomycetes are a large and variable group of microorganisms occurring in many habitats, where they take part in the degradation of organic material. Many microbial strains degrading various aromatic pollutants have been isolated from this group. Enzymes so far characterized have been reported to be monooxygenases, hydroxylases, or dioxygenases (for a review, see reference 31). Actinomycetes, including those degrading xenobiotic compounds, have also been shown to be environmentally tenacious, making them a potential source of microorganisms for bioremediation (6, 18, 29, 30). Mycobacterium fortuitum CG-2 was isolated from a tetrachloroguaiacol enrichment culture and shown to degrade several chlorinated phenols, guaiacols, and syringols at micromolar levels (15). Pentachlorophenol (PCP) was shown to be mineralized to CO2, and tetrachloro-para-hydroquinone (TeCH) was detected as an initial intermediate. We have shown that the degradation of polychlorophenols, guaiacols, and syringols proceeds through chlorinatedparahydroquinones in three different Rhodococcus chlorophenolicus strains (1, 3, 12, 13, 15) and is mediated by a membrane-associated cytochrome P-450 enzyme (28). The product of this para-hydroxylation, TeCH, is further orthohydroxylated by a soluble halohydroquinone dehalogenase (4, 14, 28). In this paper, we report on the enzymatic dehalogenation of chlorinated, brominated, and fluorinated phenols and hydroquinones by M. fortuitum CG-2 cell fractions. We show that the halogenated phenols were converted to halogenated para-hydroquinones by a membrane-associated cytochrome P-450 enzyme. The halohydroquinones formed by the para-hydroxylation were further dehalogenated by a soluble halohydroquinone dehalogenase. The results show that M. fortuitum CG-2 dehalogenases are similar to those previously described by us for R. chlorophenolicus PCP-1 and CP-2 (3, 4, 14, 15, 28).
*
MATERIALS AND METHODS Bacterial strains and culture conditions. M. fortuitum CG-2 (12, 16) was grown in nutrient broth-yeast extract medium (21) in a gyratory shaker at 28°C. Two-day-old cultures were harvested or induced by the addition of increasing amounts (10 to 50 FtM) of PCP at 24-h intervals and then harvested. Cell fractionation. Cells were harvested by centrifugation (3,000 x g, 10 min, 4°C), resuspended (1 g [wet weight] ml-') in borax buffer (50 mM, pH 8.0) (22), and washed once with the same buffer. Cells were disrupted in a French press at 700 lb/in2, DNase I (50 ,g ml-'; Boehringer Mannheim, Mannheim, Germany) was added to the cell extract, and the mixture was incubated for 1 h at 22°C. The unbroken cells were removed by centrifugation (3,000 x g, 10 min, 4°C), and the supernatant was further centrifuged at 150,000 x g for 90 min at 4°C. The supernatant contained the soluble proteins, and the pellet contained the membrane-associated proteins. The pellet was washed once with 10 volumes of borax buffer and resuspended in the original volume of the same buffer, Analyses. For activity assays, 1.0 mg of protein ml-' and 20 to 50 ,uM substrate (PCP, pentafluorophenol [PFP], pentabromophenol [PBP], TeCH, tetrafluoro-para-hydroquinone [TeFH], or tetrabromo-para-hydroquinone [TeBH]) were used. Protein was determined by the method of Bradford (5) with a reagent from Bio-Rad Laboratories, Richmond, Calif., and with egg white lysozyme (Sigma, St. Louis, Mo.) for calibration. Assays for halophenol dehalogenation were carried out in borax buffer at pH 8.0. The pH optimum of the reaction was tested with borax buffer (pH range, 5.8 to 9.2). For assays for halohydroquinone dehalogenation, ascorbic acid (final concentration, 1 mg ml-') was added to prevent abiotic oxidation of halohydroquinones. The enzymatic activity was calculated from the linear period of activity (4 h). A reaction mixture with no enzyme added served as the blank for both types of assays. Halogenated phenolic compounds were analyzed as acetylated derivatives by gas-liquid chromatography (GLC) by an internal standard method as described earlier (2, 3). To improve the acetylation efficiency of the brominated conge-
Corresponding author. 5669
5670
UOTILA ET AL.
ners, we added acetone to the acetylation buffer to 10% (2). Fluorinated congeners were extracted in pentane, and brominated and chlorinated congeners were extracted in heptane. An HP-5 fused silica capillary column (25 m by 0.2 mm; 0.33 ,um; Hewlett Packard, Palo Alto, Calif.) was used. The temperature programs used for the analysis were as follows: for TeCH and TeBH, 50°C for 1 min and 10°C/min to 300°C; for TeFH, 20°C for 1 min, 10°C/min to 60°C, and 50°C/min to 180°C. Metabolites emerging were identified by GLC-mass spectrometry (MS) as described by Apajalahti and SalkinojaSalonen (3, 4). In the analysis of para-hydroxylation products with an 1802 label, the following ions were used:
2,3,5-trichloro-para-hydroquinone (2,3,5-TCH), mle 212, 214, 216, 218, 220. Anoxic assays and isotopic labeling. For the anoxic assays, the reaction mixture was enclosed in a gas-tight vial and oxygen was removed by sequential evacuation and purging with argon (99.998%). A similarly deoxygenated substrate was injected into the vial to start the reaction, and acetylation reagents were injected to stop the reaction. In the oxygen labeling experiments, 1802 gas (isotopic purity, 80 to 90%; Msd isotopes, Montreal, Quebec, Canada) was injected into the ampoule to a partial 1802 pressure of 0.1 and the reaction was started by the injection of a deoxygenated substrate (2,3,5-trichlorophenol [2,3,5-TCP]) into the ampoule to a final concentration of 20 ,uM and stopped by the addition of acetylation reagents after 4 h of incubation at 22°C. Labeling experiments with H2180 were performed like the aerobic ones, except that the solution was made 20% with respect to H2180-labeled water (isotopic purity, 97%; Aldrich Chemical Co., Milwaukee, Wis.). The reaction was started by the addition of an anaerobic substrate solution (2,3,5-TCP; final concentration, 20 ,uM). Inhibition assays. The inhibitors tested were Tiron (5 mM; Siegfried S.A.), methylenebisthiocyanate (5 mM; Riedel-deHaen), metyrapone (5 mM; Sigma), menadione (5 mM; vitamin K3, prereduced; Sigma), parathion (5 mM; Ehrenstorfer, Augsburg, Germany) and SKF-525A (5 mM; Smith Kline & French Laboratories). Inhibition by carbon monoxide was tested at partial pressures of 1.0, 0.1, and 0.01 in gas-tight vials. Spectrophotometric analysis. Absorption spectra were measured at room temperature with the single-beam mode of a Shimadzu UV-3000 spectrophotometer. Sodium dithionite (a few grains) was added to the protein solution (2.3 mg in 0.5 ml of borax buffer) to create a reduced environment, and the spectrum (400 to 500 nm) was measured before and after purging with carbon monoxide. The concentration of cytochrome P-450 was calculated from the reduced carbon monoxide differential spectrum with the specific extinction coefficient of the Pseudomonas putida enzyme, A45049 = 92.8 cm-' mM-1 (11). RESULTS Localization of the halophenol-para-hydroxylating activity in cell extracts of M. fortuitum CG-2. From previous work, we knew that M. fortuitum CG-2 was able to degrade (mineralize) PCP via para-hydroxylation to TeCH (15). To test whether other pentahalophenols would be attacked, we tested the degradation of PFP and PBP by crude extracts of PCP-induced cells and analyzed the products formed from PEP and PBP by GLC-MS. A new peak emerged in the gas chromatogram of each halophenol after incubation with the crude extract, the retention times (the elution times, in minutes, of the model compounds TeCH, TeBH, and TeFH
J. BACTERIOL.
were 11.3, 16.1, and 8.1, respectively, and those for the
metabolites formed from these pentahalogenated phenols were the same) and the mass spectra (Fig. 1) being identical to those of authentic TeCH, TeFH, and TeBH. These results
indicate that the pentahalophenols had undergone dehalogenation and para-hydroxylation while in contact with the crude extracts of M. fortuitum CG-2. The reaction was optimal at pH 7 to 8. Outside the pH range of 6.5 to 9.0, no dehalogenation was observed. To localize the PCP-, PFP-, and PBP-para-hydroxylating activity in M. fortuitum CG-2 cells, we prepared cell-free crude extracts from the cells and separated them into a 150,000 x g supernatant (soluble proteins) and a 150,000 x g pellet (membrane-associated proteins). Dehalogenation of halophenols was tested for each fraction as described in Materials and Methods. Table 1 shows the dehalogenation activities of the extracts prepared from PCP-induced and uninduced cells of M. fortuitum CG-2 and the localization of the halophenol-consuming enzyme activity in the cell fractions. The rates of turnover of PCP, PFP, and PBP in crude extracts from PCP-induced cells ranged from 1.7 to 2 nmol h-1 mg of protein-1 (Table 1). Crude extracts prepared from uninduced cells showed no detectable halophenol-degrading enzyme activity, indicating that this enzyme activity was inducible. Treatment of the crude extracts with proteinase K (1 mg ml-') destroyed the halophenol-para-hydroxylating activity, showing that it was mediated by enzyme protein. The induction factor for the halophenol dehalogenases was ca. 40-fold in M. fortuitum CG-2. When the cell extracts were fractionated by centrifugation (150,000 x g), 67% of the halophenol-degrading enzyme activity of the crude extracts was recovered in the 150,000 x g pellet, which contained 33% of the total cell extract protein (Table 1). No halophenol-para-hydroxylating activity was found in the 150,000 x g supernatant. Phenol was not degraded (
